Embryo Genetic Testing (PGD)

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1 Embryo enetic esting (PD) hromosome Screening Fact Sheet What are chromosomes and why are they important in fertility? n individual s genetic information is packaged into strings of DN called chromosomes. Normal individuals contain 46 chromosomes, which are arranged in 23 chromosome pairs. hese chromosome pairs are labelled 1 to 22 (the autosomes) and X and Y (the sex chromosomes). Females carry two X chromosomes, whereas males carry an X and a Y chromosome (refer to Figure 1). Figure 1: n example of a set of female chromosomes. Unlike the other cells in the body, egg and sperm cells only contain 23 individual chromosomes, one of each of the 22 autosomes and one of the sex chromosomes (either X or Y). When the egg and sperm unite during conception, these chromosomes combine. his results in the formation of an embryo with the usual 46 chromosomes, arranged in pairs. Errors in cell division in the developing egg, sperm or embryo can result in the formation of embryos with an abnormal number of chromosomes (ie: missing or extra chromosome/s). his is known as chromosomal aneuploidy. Embryos with a chromosomal aneuploidy will fail to implant, miscarry, or result in the birth of an affected child (eg: a child with Down syndrome). pproximately 50% of spontaneous abortions are caused by chromosomal aneuploidy. How common are chromosome abnormalities in embryos? It is well documented that the frequency of chromosomal abnormalities in embryos increases with maternal age. herefore, older women will be less likely to obtain a normal embryo for transfer compared with younger women (refer to Figure 2). However, once a normal embryo is identified for transfer, the pregnancy rate in older women is not significantly different from that of younger women.

2 Figure 2: Percentage of normal embryos on Day 3 versus Day 5 of embryo development Normal embryos (%) Day 3 Day Maternal ge What is chromosome screening? Preimplantation enetic Diagnosis (PD) with chromosome screening can be used to screen IVF embryos for chromosome abnormalities prior to implantation. Only embryos that have the correct number of chromosomes will be selected for transfer to the uterus. his testing will help to ensure that the embryo that is selected for transfer has the best possible chance of developing into a healthy baby. raditionally, PD tests have only been able to analyse a limited number of chromosomes at a time. his has restricted the amount of genetic information that can be obtained from embryos and has therefore limited the diagnostic capability and benefits of PD. Newer PD technologies, such as array-h and SNP arrays, are capable of simultaneously analysing all human chromosomes in a single test. hese technologies have enabled IVF clinics to more confidently predict whether an individual embryo is chromosomally normal, resulting in the transfer of embryos that have a higher chance of implantation and development to term. Other PD techniques that only test some of the chromosomes are at risk of missing important genetic information. Since each embryo is precious, accuracy is critical to avoid discarding normal embryos or transferring abnormal embryos. How is chromosome screening done? Step 1: enetic counselling in PD clinic Prior to the commencement of an IVF cycle, the couple attend an appointment with a genetic counsellor. During this appointment the couple will be provided with information relating to PD and will have an opportunity to have any questions answered. his appointment is free of charge and enables couples to decide if PD is the right option for them. Step 2: IVF and Embryo biopsy ll couples undergoing chromosome screening must undertake an IVF cycle to stimulate the woman s ovaries to produce a number of eggs. hese eggs are collected and fertilised using the male partner s sperm. he resulting embryos are cultured in the laboratory and their growth is monitored on a daily basis. Embryo biopsy (sampling cell/s from the embryo for genetic testing) can be performed at two different stages during an embryo s development: 1. Day 3 embryo biopsy is performed 3 days after fertilization, when the embryo is at the cleavage stage and is typically composed of 6 to 8 cells. Embryos that have developed to at least 5 cells on Day 3 are suitable for biopsy. hole is drilled in the outer shell of the embryo and 1 or 2 cells are removed for genetic analysis (refer to Figure 3).

3 Figure 3: Day 3 embryo biopsy 2. Day 5/6 embryo biopsy is performed 5 or 6 days after fertilization. By this time, the embryo should have developed to the blastocyst stage, and should be comprised of an inner cell mass (which will go on to form the fetus) and trophectoderm cells (which will go on to form the placenta). Embryos need to have a clear inner cell mass and a suitable number of healthy trophectoderm cells to be considered suitable for biopsy. pproximately 5 trophectoderm cells are removed for genetic analysis (Refer to Figure 4). Figure 4: Day 5/6 embryo biopsy In the majority of cases, Monash IVF recommends Day 5/6 biopsy. his is due to the following reasons: Randomised control trials have shown that Day 5/6 biopsy is better for the embryo compared with Day 3 biopsy (Scott et al, 2013). he embryo has more cells on Day 5/6 of development (~100 to 150 cells) compared with Day 3 of development (~6 to 8 cells). his means: - More cells can be biopsied for genetic testing on Day 5/6 of development compared with Day 3 of development (ie: approximately 5 cells versus 1-2 cells, respectively). he availability of more cells improves the accuracy of the PD test results. - Despite biopsying more cells from Day 5/6 embryos, a smaller percentage of cells is removed from the embryo following a Day 5/6 biopsy compared with a Day 3 biopsy (ie: we are removing approximately 5/100 (5%) cells on Day 5/6, compared with 1/6 (17%) cells on Day 3). Day 5/6 embryos are more likely to be chromosomally normal then Day 3 embryos (as some of the embryos that are abnormal on Day 3 do not have the developmental capacity to grow to Day 5/6 in culture). herefore, growing the embryos to Day 5/6 before performing the PD testing provides an element of natural selection. Day 5/6 embryo biopsy enables the patient to confirm that their embryos are capable of developing to an advanced stage in culture before proceeding with PD testing. he alternative is to perform PD testing on Day 3 with the knowledge that the embryos may not continue to develop and therefore may not be suitable for transfer from an Embryology perspective. Step 3: enetic testing he biopsied cells are transferred to a small test tube for genetic testing. Monash IVF currently offers chromosome screening using two different test types, called rray-h and SNP array testing. More information on each of these test types is included below: 1. rray-h (performed using 24Sure technology) In this procedure, the DN from the embryonic cells is multiplied thousands of times (to generate enough DN for testing) and is then fluorescently labelled. Once labelled, the DN sample is placed on a microarray platform. his microarray platform consists of a matrix of thousands of different DN probes which are specific to each chromosome. he fluorescently labelled DN from the embryo biopsy sample binds to the DN probes on the microarray platform. By comparing the fluorescent intensity of the embryo biopsy sample with that of a control male and

4 control female sample, it is biopsied cell/s (Figure 5). Figure 5: rray-h procedure possible to determine the copy number of each chromosome in the est cell/s + est DN 46,XY (male) ontrol DN 46,XX (female) ontrol DN Loss of fluorescence relative to controls indicates the embryo is missing a chromosome Same fluorescence as controls indicates the embryo has the normal number of chromosomes ain of fluorescence relative to controls indicates the embryo has an extra chromosome 2. SNP array testing (performed using parental support ) In this procedure, the DN from the embryonic cells is multiplied thousands of times (to generate enough DN for testing) and is placed on a microarray platform. his microarray platform contains probes for over 300,000 different DN sites. he DN from the embryo biopsy sample binds to the DN probes on the microarray platform. Following binding, it is possible to read the DN code at each of these DN sites. By screening a blood sample from each partner in parallel with the embryonic cells, it is possible to determine which chromosome/ /s the embryo inherited from each parent (Figure 6). In addition to determining chromosome copy number, this testing is capable of identifying the source of any additional or missing chromosomes (ie: maternal or paternal origin). his information may be useful to assist the patient with future reproductive decision making. Figure 6: PD testing method using SNP arrays. est cell/s + est DN DN from male partner DN from female partner hromosomes inherited by embryo Step 4: Embryo transfer Because of the time taken to perform the genetic testing, the embryos must be frozen following biopsy. Final results are usually available 2 to 3 weeks after biopsy. If available, one or two normal embryos can be thawed for use in a frozen embryo transfer cycle. PD scientist/embryologist will discuss the PD results with the patient prior to transfer. he patient s IVF nurse will organise a pregnancy test to be performed on Day 16 of the frozen embryo transfer cycle. his process should

5 increase the chance of a successful pregnancy and significantly reduce the risk of miscarriage. Surplus normal embryos will remain in storage. hese embryos may be used in a subsequent cycle. hromosomally abnormal embryos will be removed from storage and allowed to succumb. What type of chromosome screening is best for me? here are differences in the testing capabilities of array-h compared with SNP arrays. SNP array testing (with parental support) has the diagnostic advantage of being capable of detecting a wider range of chromosome abnormalities than array-h (able 1). However, as array-h is charged on a per embryo basis, this testing may appeal to patients with small embryo numbers who would otherwise elect not to proceed with 24 chromosome screening. enetic counselling is an important step to help patients understand the differences between these two types, so that they make an informed decision regarding which test type is best in their case. he PD testing option that is performed will be decided by the patient in consultation with their IVF specialist and enetic ounsellor. able 1: Detection capabilities of array-h compared with SNP arrays. riterion rray-h SNP arrays Screen 24 chromosomes Detect haploidy (a missing set of all chromosomes, which results in implantation failure) Detect triploidy (an extra set of all chromosomes, which results in implantation failure or miscarriage) Detect uniparental disomy (the presence of two copies of a given chromosome from one parent and none from the other. his can result in particular genetic syndromes or medical, cognitive or physical disabilities). Some Some x Determine the parental origin of chromosome abnormalities x onfirm genetic parentage x Detect extraneous DN contamination x Why choose Monash IVF for PD? Monash IVF has offered PD as a clinical service since 1996 and is one of the few centres in ustralia that specialises in this area of reproductive medicine. In 1996 we were proud to report the birth of ustralia s first PD babies and since then we have performed over 3,000 PD cycles with proven high success rates. Our specialised genetics team contains highly qualified experts in PD, ensuring the best quality of care for patients. he genetics team at Monash IVF is responsible for providing a specialised PD service not only to our own patients, but also to patients undergoing IVF cycles at fourteen different IVF clinics throughout ustralia and New Zealand. While the main PD laboratory is located in layton, Melbourne, ustralia, embryo biopsy can be performed away from the genetics laboratory and the embryonic cells sent by courier to layton. entralising the genetic testing enables patients to access the highest levels of expertise without having to leave their home state. Where can I get more information? If you would like further information regarding the PD program at Monash IVF, please feel free to contact a member of the enetics team on